Although <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) protects against experimental acute lung injury, the mechanisms for the protective effect are incompletely understood. Therefore, the time-dependent effects of KGF on alveolar epithelial fluid transport were studied in rats 48-<em>2</em>40 h after intratracheal administration of KGF (5 mg/kg). There was a marked proliferative response to KGF, measured both by in vivo bromodeoxyuridine staining and by staining with an antibody to a type II cell antigen. In controls, alveolar liquid clearance (ALC) was <em>2</em>3 +/- 3%/h. After KGF pretreatment, ALC was significantly increased to 30 +/- <em>2</em>%/h at 48 h, to 39 +/- <em>2</em>%/h at 7<em>2</em> h, and to 36 +/- 3%/h at 1<em>2</em>0 h compared with controls (P < 0.05). By <em>2</em>40 h, ALC had returned to near-control levels (<em>2</em>6 +/- <em>2</em>%/h). The increase in ALC was explained primarily by the proliferation of alveolar type II cells, since there was a good correlation between the number of alveolar type II cells and the increase in ALC (r = 0.9<em>2</em>, P = 0.0<em>2</em>). The fraction of ALC inhibited by amiloride was similar in control rats (33%) as in 7<em>2</em>-h KGF-pretreated rats (38%), indicating that there was probably no major change in the apical pathways for Na uptake in the KGF-pretreated rats at this time point. However, more rapid ALC at 1<em>2</em>0 h, compared with 48 h after KGF treatment, may be explained by greater maturation of alpha-epithelial Na channel, since its expression was greater at 1<em>2</em>0 than at 48 h, whereas the number of type II cells was the same at these two time points. beta-Adrenergic stimulation with terbutaline 7<em>2</em> h after KGF pretreatment further increased ALC to 50 +/- 7%/h (P < 0.5). In summary, KGF induced a sustained increase over 1<em>2</em>0 h in the fluid transport capacity of the alveolar epithelium. This impressive upregulation in fluid transport was further enhanced with beta-adrenergic agonist therapy, thus providing evidence that two different treatments can simultaneously increase the fluid transport capacity of the alveolar epithelium.

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and <em>2</em>% fetal bovine serum (FBS; KGF/<em>2</em>) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx3<em>2</em> and Cx<em>2</em>6 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx3<em>2</em>, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/<em>2</em> retained expression of Cx3<em>2</em> and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx3<em>2</em> and Cx43 were at the plasma membrane of cells grown in KGF/<em>2</em>, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed approximately 3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in <em>2</em>% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.

Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity. N(6)-furfuryladenosine (FAdo, kinetin-riboside) displays anti-proliferative and apoptogenic activity against various human cancer cell lines, and FAdo has recently been shown to suppress tumor <em>growth</em> in murine xenograft models of human leukemia and melanoma. In this study, FAdo-induced genotoxicity, stress response gene expression, and cellular ATP depletion were examined as early molecular consequences of FAdo exposure in MiaPaCa-<em>2</em> pancreas carcinoma, A375 melanoma, and other human cancer cell lines. FAdo, but not adenosine or N(6)-furfuryladenine (FA), displayed potent anti-proliferative activity that was also observed in human primary fibroblasts and <em>keratinocytes</em>. Remarkably, massive ATP depletion and induction of genotoxic stress as assessed by the alkaline comet assay occurred within 60-180min of exposure to low micromolar concentrations of FAdo. This was followed by rapid upregulation of CDKN1A and other DNA damage/stress response genes (HMOX1, DDIT3, and GADD45A) as revealed by expression array and Western analysis. Pharmacological and siRNA-based genetic inhibition of adenosine kinase (ADK) suppressed FAdo cytotoxicity and also prevented ATP depletion and p<em>2</em>1 upregulation suggesting the importance of bioconversion of FAdo into the nucleotide form required for drug action. Taken together our data suggest that early induction of genotoxicity and energy crisis are important causative <em>factors</em> involved in FAdo cytotoxicity.

OBJECTIVE

The signals that govern the upregulation of nutrient absorption (adaptation) after intestinal resection are not well understood. A Gastric Roux-en-Y bypass (GRYB) model was used to isolate the relative contributions of direct mucosal stimulation by nutrients, biliary-pancreatic secretions, and systemic enteric hormones on intestinal adaptation in short bowel syndrome.

METHODS

Male rats (350-400 g; n = 8/group) underwent sham or GRYB with pair feeding and were observed for 14 days. Weight and serum hormonal levels (glucagon-like peptide-<em>2</em> [GLP-<em>2</em>], PYY) were quantified. Adaptation was assessed by intestinal morphology and crypt cell kinetics in each intestinal limb of the bypass and the equivalent points in the sham intestine. Mucosal <em>growth</em> <em>factors</em> and expression of transporter proteins were measured in each limb of the model.

RESULTS

The GRYB animals lost weight compared to controls and exhibited significant adaptive changes with increased bowel width, villus height, crypt depth, and proliferation indices in the alimentary and common intestinal limbs. Although the biliary limb did not adapt at the mucosa, it did show an increased bowel width and crypt cell proliferation rate. The bypass animals had elevated levels of systemic PYY and GLP-<em>2</em>. At the mucosal level, insulin-like <em>growth</em> factor-1 (IGF-1) and basic fibroblast <em>growth</em> factor (bFGF) increased in all limbs of the bypass animals, whereas keratinocyte <em>growth</em> factor (KGF) and epidermal <em>growth</em> factor (EGF) had variable responses. The expression of the passive transporter of glucose, GLUT-<em>2</em>, expression was increased, whereas GLUT-5 was unchanged in all limbs of the bypass groups. Expression of the active mucosal transporter of glucose, SGLT-1 was decreased in the alimentary limb.

CONCLUSIONS

Adaptation occurred maximally in intestinal segments stimulated by nutrients. Partial adaptation in the biliary limb may reflect the effects of systemic hormones. Mucosal content of IGF-1, bFGF, and EGF appear to be stimulated by systemic hormones, potentially GLP-<em>2</em>, whereas KGF may be locally regulated. Further studies to examine the relationships between the <em>factors</em> controlling nutrient-induced adaptation are suggested. Direct contact with nutrients appears to be the most potent factor in inducing mucosal adaptation.

Microvesicles (MVs) derived from human mesenchymal stem cells (MSC MVs) were demonstrated to ameliorate inflammation in lungs. We have found their content of mRNA for <em>keratinocyte</em> <em>growth</em> <em>factor</em> was partly involved in their therapeutic effects. As MSC MVs also contained a substantial quantity of angiopoietin-1 (Ang-1) mRNA, which plays an essential role in vascular stabilization and resolving inflammation, we hypothesized that Ang-1 mRNA might similarly account for a part of their therapeutic effects. We downregulated Ang-1 mRNA expression in MVs, using a lentivirus vector carrying Ang-1 short hairpin RNA to transfect MSCs. A mouse model of lipopolysaccharide induced acute lung injury (ALI) was used in vivo. We also studied in vitro interactions between Ang-1 mRNA deficient MVs on macrophages and human lung microvascular endothelial cells. Compared with negative control, Ang-1 mRNA deficient MVs increased the influx of neutrophils and macrophage inflammatory protein-<em>2</em> levels in bronchoalveolar lavage fluid by 136% and 105%, respectively, suggesting a deteriorative lung inflammation and a failure to restore pulmonary capillary permeability assessed by Evan's blue dye and bronchoalveolar lavage albumin level. In vitro, the addition of Ang-1 mRNA deficient MVs failed to maintain the integrity of endotoxin-stimulated microvascular endothelial cells and abrogated the decrease in tumor necrosis <em>factor</em>-α level and the increase in interleukin-10 level mediated by negative control in RAW <em>2</em>64.7 cells. In summary, the therapeutic effects of MVs in ALI, and their immunomodulatory properties on macrophages were partly mediated through their content of Ang-1 mRNA. Stem Cells <em>2</em>017;35:1849-1859.

All-trans retinoic acid (RA) compromises epidermal differentiation and causes <em>keratinocyte</em> hyperproliferation through mechanisms not completely understood, but may involve the regulatory matrix molecule hyaluronan. In this work, the influences of all-trans RA on epidermal morphology and hyaluronan metabolism were examined in organotypic and monolayer cultures of rat epidermal <em>keratinocytes</em> (REKs). All-trans RA treatment of organotypic REK cultures (10 days) increased the synthesis of hyaluronan, the expression of hyaluronan synthases Has<em>2</em> and Has3, and the CD44 receptor, with hyperplasia of the epidermis. The hyperplasia and hyaluronan production induced by all-trans RA were blocked with (1) AG1478, an inhibitor of the EGFR; (<em>2</em>) UO1<em>2</em>6, an inhibitor of the MAPK/ERK kinase, and (3) GM6001, an inhibitor of the matrix metalloproteinases. These effects were consistent with the findings that all-trans RA upregulated heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em> mRNA expression and increased the phosphorylation of EGFR and extracellular signal-regulated kinase 1/<em>2</em> (ERK1/<em>2</em>). Interestingly, the activation of EGFR and ERK1/<em>2</em> was seen already 30 minutes after all-trans RA treatment, suggesting that the activation of this signaling pathway is a primary response to all-trans RA. These results indicate that the effects of all-trans RA on <em>keratinocyte</em> proliferation and hyaluronan synthesis are partly mediated through EGFR signaling.

The effects of laser phototherapy on the release of <em>growth</em> <em>factors</em> by human gingival fibroblasts were studied in vitro. Cells from a primary culture were irradiated twice (6 h interval), with continuous diode laser [gallium-aluminum-arsenium (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP),_660 nm] in punctual and contact mode, 40 mW, spot size 0.04<em>2</em> cm(<em>2</em>), 3 J/cm(<em>2</em>) and 5 J/cm(<em>2</em>) (3 s and 5 s, respectively). Positive [10% fetal bovine serum (FBS)] and negative (1%FBS) controls were not irradiated. Production of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and basic fibroblast <em>growth</em> <em>factor</em> (bFGF) was quantified by enzyme-linked immunosorbent assay (ELISA). The data were statistically compared by analysis of variance (ANOVA) followed by Tukey's test (P </= 0.05). The characterization of the cell line indicated a mesenchymal nature. KGF release was similar in all groups, while that of bFGF was significantly greater (1.49-times) in groups treated with infra-red laser. It was concluded that increased production of bFGF could be one of the mechanisms by which infra-red laser stimulates wound healing.

Our aim was to evaluate (a) whether there is differential expression of the endothelial regulator vascular endothelial <em>growth</em> <em>factor</em> (VEGF), its receptor (VEGFR-<em>2</em>), and the hypoxia-associated glucose transporter molecule, GLUT-1, in skin biopsies from different disease subtypes of systemic sclerosis (SSc) and (b) whether they associate with dermal calcinosis, a significant complication of SSc. Skin punch biopsies were taken from the forearms of 66 SSc patients including 18 with limited cutaneous disease without calcinosis (lcSSc), <em>2</em>3 with calcinosis (lcSSc/cal), and <em>2</em>5 with diffuse cutaneous disease (dcSSc) and from 1<em>2</em> healthy control subjects. The histological appearance of the skin was graded as G0 (normal), G1 (dermal edema), or G<em>2</em> or G3 (increasing fibrotic changes). Immunohistochemistry was performed with antibodies to VEGF, VEGFR-<em>2</em>, and GLUT-1. Staining was assessed in the epidermis, microvessels, and fibroblasts. The Kruskal-Wallis 1-way analysis of variance was used to compare the data between disease groups. VEGF protein was located in the epidermis and in dermal endothelial cells, pericytes, fibroblasts, and inflammatory cells. In dcSSc only, there was a significant increase in VEGF staining intensity in the <em>keratinocytes</em> and pericytes and the lowest percentage of microvessels with VEGF-positive endothelial cells. GLUT-1 protein was located in the epidermis, erythrocytes, and perineurium. In both lcSSc/cal and dcSSC, but not lcSSc, there were significant increases in GLUT-1 staining intensity of <em>keratinocytes</em>. We propose that in patients with dcSSc, there is a net increase in unbound VEGF in skin that may account for the raised levels of VEGF in serum reported by others. Increased GLUT-1 expression in lcSSc/cal and dcSSc indicates that hypoxia is an associated <em>factor</em>.

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type <em>2</em> alveolar epithelial cells (AEC<em>2</em>). Postulating that apoptosis might occur in AEC<em>2</em> after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC<em>2</em> from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p<em>2</em>1, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC<em>2</em> cultured from hyperoxic rats when compared with control AEC<em>2</em>. In contrast, significant apoptosis was not detected in freshly isolated AEC<em>2</em> from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC<em>2</em> damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of <em>2</em>0 ng/ml of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p<em>2</em>1, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC<em>2</em>, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival <em>factor</em> in AEC<em>2</em> by limiting apoptosis in the lung after acute hyperoxic injury.

The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear <em>factor</em>-E<em>2</em>-related <em>factor</em> <em>2</em> (NRF<em>2</em>)-mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf<em>2</em>(-/-) mice have delayed wound closure rates compared with Nrf<em>2</em>(+/+) mice, which is, at least partially, due to greater oxidative DNA damage, low transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF<em>2</em> pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized <em>keratinocyte</em> cells confirm that NRF<em>2</em> contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF<em>2</em> in diabetic wound healing and the therapeutic benefits of activating NRF<em>2</em> in this disease, laying the foundation for future clinical trials using NRF<em>2</em> activators in treating diabetic skin ulcers.

Inhibition of apoptosis plays an important role in the cellular immortalization and transformation induced by E6 and E7 oncoproteins of human papillomavirus (HPV). Here, we report that the transcription of the inhibitor of apoptosis gene, cellular inhibitor of apoptosis protein <em>2</em>, (c-IAP<em>2</em>), is significantly upregulated in HPV16 E6/E7-immortalized human oral <em>keratinocytes</em> (HOK16E6E7). Overexpression of E6/E7 from the high-risk HPV16 or 18, but not from the low-risk HPV6, activated c-IAP<em>2</em> promoter. E6 from HPV16 and 18 played a major role in the activation. In addition, the induction of c-IAP<em>2</em> transcription required nuclear <em>factor</em>-kappaB activity. Overexpression of c-IAP<em>2</em> in normal human oral <em>keratinocyte</em> conferred resistance to tumor necrosis <em>factor</em>-alpha (TNF-alpha)/cycloheximide (CHX)-induced apoptosis, suggesting the increased c-IAP<em>2</em> expression in HOK16E6E7 may protect the cells from TNF-alpha-mediated cell death. Moreover, depletion of endogenous c-IAP<em>2</em> using RNA interference in HOK16E6E7 induced apoptosis, indicating that c-IAP<em>2</em> is necessary for HPV16 E6/E7-induced resistance to apoptosis and cell survival. Of note, high levels of c-IAP<em>2</em> transcription were found in several HPV16- or HPV18-positive cancer cells, and depletion of c-IAP<em>2</em> caused cell death in HPV18-positive HeLa cells. Thus, upregulation of c-IAP<em>2</em> by E6 and E7 may confer resistance to apoptosis that is necessary for sustained <em>growth</em> of some HPV16- and HPV18-positive cancer cells.

Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular <em>keratinocytes</em> throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both <em>factors</em> in the <em>growth</em> of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular <em>keratinocytes</em> involves the up-regulation of the cyclooxygenase <em>2</em> enzyme by a mesenchymal paracrine <em>factor</em>, the hepatocyte <em>growth</em> <em>factor</em>. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects <em>keratinocytes</em> from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair <em>growth</em>.

We have shown that autocrine proliferation of human <em>keratinocytes</em> (KCs) is strongly dependent upon amphiregulin (AREG), whereas blockade of heparin-binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) inhibits KC migration in scratch wound assays. Here we demonstrate that expression of soluble HB-EGF (sHB-EGF) or full-length transmembrane HB-EGF (proHB-EGF), but not proAREG, results in profound increases in KC migration and invasiveness in monolayer culture. Coincident with these changes, HB-EGF significantly decreases mRNA expression of several epithelial markers including keratins 1, 5, 10, and 14 while increasing expression of markers of cellular motility including SNAI1, ZEB1, COX-<em>2</em>, and MMP1. Immunostaining revealed HB-EGF-induced expression of the mesenchymal protein vimentin and decreased expression of E-cadherin, as well as nuclear translocation of β-catenin. Suggestive of a trade-off between KC motility and proliferation, overexpression of HB-EGF also reduced KC <em>growth</em> by >90%. We also show that HB-EGF is strongly induced in regenerating epidermis after partial-thickness wounding of human skin. Taken together, our data suggest that expression of HB-EGF in human KCs triggers a migratory and invasive phenotype with many features of epithelial-mesenchymal transition (EMT), which may be beneficial in the context of cutaneous wound healing.

BACKGROUND

A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed.

RESULTS

By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes.

CONCLUSIONS

Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype. See: http://www.biomedcental.com/1741-7007/8/125.

There are 600 million betel quid (BQ) chewers in the world. BQ chewing is a major etiologic <em>factor</em> of oral cancer. Areca nut (AN) and arecoline may inhibit the <em>growth</em> of oral mucosal fibroblasts (OMF) and <em>keratinocytes</em>. In this study, AN extract (100-800 microg/ml) and arecoline (<em>2</em>0-1<em>2</em>0 microM) inhibited the <em>growth</em> of oral KB cells by 36-90 and 15-75%, respectively. Exposure to arecoline >> 0.<em>2</em> mM) for <em>2</em>4 h induced G(<em>2</em>)/M cell cycle arrest of OMF and KB cells. Areca nut extract >> 400 microg/ml) also induced G(<em>2</em>)/M arrest of KB cells, being preceded by S-phase arrest at 7-h of exposure. No evident sub-G(0)/G(1) peak was noted. Marked retraction and intracellular vacuoles formation of OMF and KB cells were observed. Glutathione (GSH) level, mitochondrial membrane potential (Deltabetam) and H(<em>2</em>)O(<em>2</em>) production of KB cells were measured by flow cytometry. GSH level [indicated by 5-chloromethyl-fluorescein (CMF) fluorescence] was depleted by <em>2</em>4-h exposure of KB cells to arecoline (0.4-1.<em>2</em> mM) and AN extract (800-1<em>2</em>00 microg/ml), with increasing the percentage of cells in low CMF fluorescence. By contrast, arecoline (0.1-1.<em>2</em> mM) and AN extract (800-1<em>2</em>00 microg/ml) induced decreasing and increasing H(<em>2</em>)O(<em>2</em>) production (by <em>2</em>',7'-dichloro- fluorescein fluorescence), respectively. Hyperpolarization of Deltabetam (increasing of rhodamine uptake) was noted by <em>2</em>4-h exposure of KB cells to arecoline (0.4-1.<em>2</em> mM) and AN extract (800-1<em>2</em>00 microg/ml). AN extract (100- 1<em>2</em>00 microg/ml) and arecoline (0.1-1.<em>2</em> mM) induced little DNA fragmentation on KB cells within <em>2</em>4 h. These results indicate that AN ingredients are crucial in the pathogenesis of oral submucous fibrosis (OSF) and oral cancer by differentially inducing the dysregulation of cell cycle control, Deltabetam, GSH level and intracellular H(<em>2</em>)O(<em>2</em>) production, these events being not coupled with cellular apoptosis.

Patients with oral squamous cell carcinoma (OSCC) have severe defects in antitumor immune function. Endothelial cells are potential regulators of immune cell function and have therefore been examined to determine their role in tumor-induced immune suppression. The present studies demonstrated that supernatants from endothelial cells exposed to OSCC-conditioned media (endo(OSCC-sup)) exhibited elevated levels of the immune suppressive products prostaglandin E(<em>2</em>) (PGE(<em>2</em>)) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) compared with supernatants from endothelial cells treated with medium alone (endo(medium)) or with <em>keratinocyte</em>-conditioned medium (endo(ker-sup)). Antibody neutralization of OSCC-derived VEGF prevented tumor-conditioned media from inducing endothelial cells to increase production of PGE(<em>2</em>)and VEGF. Furthermore, treatment of T-cells with supernatants from endo(OSCC-sup) resulted in diminished T-cell proliferation and decreased interferon-gamma (IFN-gamma) production compared with T-cells treated with medium or supernatants from endo(medium) or endo(ker-sup) controls. T-cell levels of granzyme B and perforin were reduced after treatment with supernatant from endo(OSCC-sup) compared with control treatments. The addition of VEGF neutralizing antibody to the OSCC-conditioned medium prevented endothelial cells from being skewed to downregulate T-cell proliferation and production of IFN-gamma, perforin, and granzyme B. Taken together, these studies provide support for the use of VEGF-targeting therapies as an immunotherapeutic agent to block induction of immune suppressive endothelial cells in patients with OSCC.

The stromelysin-<em>2</em> (SL-<em>2</em>) gene is transcriptionally active in normal human <em>keratinocytes</em> and encodes a secreted, catalytically competent but latent matrix metalloproteinase. Phorbolester induction resulted in the emergence of SL-<em>2</em> (but not SL-1 transcripts), whereas the opposite was true for human mucosal fibroblasts. Expression of <em>keratinocyte</em> SL-<em>2</em> was also induced by the two <em>keratinocyte</em> <em>growth</em> <em>factors</em>, transforming <em>growth</em> <em>factor</em>-alpha and epidermal <em>growth</em> <em>factor</em>, by the proinflammatory cytokine, tumor necrosis <em>factor</em>-alpha, but, somewhat surprisingly, not by interleukin-1 beta. The latent SL-<em>2</em> proenzyme was isolated from 1<em>2</em>-O-tetradecanoylphorbol-13-acetate-induced <em>keratinocytes</em> by immunoaffinity chromatography using a cross-reactive antibody raised against human SL-1. This procedure led to the recovery of a single M(r) 54,000 molecular species at a level of approximately 0.<em>2</em> microgram/ml of culture medium. Amino-terminal sequencing identified the protein as SL-<em>2</em> and verified the predicted signal sequence cleavage site. Conformational activation of latent SL-<em>2</em> precursor by SDS gave rise to a full-length, uncleaved (M(r) 54,000) active form and at the same time exposed a cryptic thiol group. By contrast, organomercurial activation resulted in autolytic truncation of the molecule with loss of M(r) approximately 10,000 propeptide. SL-<em>2</em> shared with (human fibroblast) SL-1 the ability to cleave casein, to "superactivate" fibroblast type procollagenase, and to form apparently binary, SDS-resistant complexes with tissue inhibitor of metalloproteinases-1.

BACKGROUND

Nonhealing ulcers affect patient quality of life and impose a substantial financial burden on the health care system.

OBJECTIVE

To systematically evaluate benefits and harms of advanced wound care therapies for nonhealing diabetic, venous, and arterial ulcers.

METHODS

MEDLINE (1995 to June 2013), the Cochrane Library, and reference lists.

METHODS

English-language randomized trials reporting ulcer healing or time to complete healing in adults with nonhealing ulcers treated with advanced therapies.

METHODS

Study characteristics, outcomes, adverse events, study quality, and strength of evidence were extracted by trained researchers and confirmed by the principal investigator.

RESULTS

For diabetic ulcers, 35 trials (9 therapies) met eligibility criteria. There was moderate-strength evidence for improved healing with a biological skin equivalent (relative risk [RR], 1.58 [95% CI, 1.20 to 2.08]) and negative pressure wound therapy (RR, 1.49 [CI, 1.11 to 2.01]) compared with standard care and low-strength evidence for platelet-derived growth factors and silver cream compared with standard care. For venous ulcers, 20 trials (9 therapies) met eligibility criteria. There was moderate-strength evidence for improved healing with keratinocyte therapy (RR, 1.57 [CI, 1.16 to 2.11]) compared with standard care and low-strength evidence for biological dressing and a biological skin equivalent compared with standard care. One small trial of arterial ulcers reported improved healing with a biological skin equivalent compared with standard care. Overall, strength of evidence was low for ulcer healing and low or insufficient for time to complete healing.

CONCLUSIONS

Only studies of products approved by the U.S. Food and Drug Administration were reviewed. Studies were predominantly of fair or poor quality. Few trials compared 2 advanced therapies.

CONCLUSIONS

Compared with standard care, some advanced wound care therapies may improve the proportion of ulcers healed and reduce time to healing, although evidence is limited.

BACKGROUND

Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Quality Enhancement Research Initiative.

Apoptosis of alveolar type II (ATII) cells in response to high-amplitude mechanical stretch represents an important mechanism of ventilation-induced lung injury. Previously, it was demonstrated in an in vitro model that stretch-induced ATII cell apoptosis was prevented by angiotensin-converting enzyme (ACE) inhibitors. This study investigates the mechanism by which ACE inhibitors prevent stretch-induced apoptosis and elucidates the role of bradykinin as an endogenous anti-apoptotic <em>factor</em>. Rat ATII cells cultured on flexible membranes were subjected to cyclic stretch (40 cycles/min; 30% increase in surface area) and compared with static controls. Angiotensinogen, the bradykinin precursor T-kininogen, and bradykinin receptor expression were measured by RT-PCR; Angiotensin II and phosphoinositol 3 OH-kinase (PI3K) activity (as phospho-Akt) were measured by enzyme-linked immunosorbent assay; and Bcl-<em>2</em> and Bcl-X(L) were measured by Western blot. Stretch did not influence angiotensinogen expression or induce angiotensin II generation. The angiotensin II receptor antagonist saralasin did not prevent stretch-induced apoptosis, whereas ACE inhibitors did. Stretch reduced ATII cell bradykinin release (T-kininogen expression and bradykinin supernatant concentration), and subsequently led to reduced PI3K activity and decreased concentrations of the anti-apoptotic proteins Bcl-<em>2</em>/Bcl-X(L). Bradykinin substitution or addition of <em>keratinocyte</em> or hepatocyte <em>growth</em> <em>factor</em> prevented stretch-induced decrease in PI3K activity and Bcl-<em>2</em>/Bcl-X(L) and reduced stretch-induced apoptosis. Mechanical stretch impairs a constitutively expressed, autocrine anti-apoptotic ATII cell survival signal involving bradykinin-mediated stimulation of the PI3K-Akt-Bcl-<em>2</em>/Bcl-X(L) pathway. Restoration of this pathway prevents stretch-induced apoptosis. This may be beneficial when mechanical ventilation cannot completely avoid alveolar overdistension to maintain oxygenation.

Normal and immortalized <em>keratinocytes</em> demonstrate large aggregates of lipid rafts, detectable by membrane staining with fluorescently tagged cholera toxin (CTx). As lipid rafts are known to regulate the function of many surface receptors, we wished to investigate their impact on the EGFR in HaCaT cells. When rafts were disrupted by cholesterol sequestration with methyl-beta-cyclodextrin (MbetaCD) or filipin III, EGFR rearranged into approximately micrometer large clusters outside the CTx(bright) raft aggregates. These clusters contained high concentrations of activated, tyrosine-phosphorylated EGFR exhibiting greatly reduced mobility in the fluorescence recovery after photobleaching experiments. EGFR activation led to the stimulation of extracellular signal-regulated kinase <em>2</em>, the phosphorylated form of which translocated to the nucleus and stimulated <em>growth</em> of the MbetaCD-treated cells. Experiments with the specific antagonistic antibody proved that the activation of EGFR by lipid raft disruption occurred without the participation of the ligand. We hypothesize that cholesterol depletion leads to the release of EGFR from the damaged rafts into the small confined areas of the membrane, where the receptor molecules are likely to be spontaneously activated owing to a very high density and/or separation from the inhibitory <em>factors</em> remaining in the surrounding portions of the membrane.

Currents that leak out of wounds generate electric fields lateral to the wound. These fields induce directional locomotion of human <em>keratinocytes</em> in vitro and may promote wound healing in vivo. We have examined the effects of <em>growth</em> <em>factors</em> and calcium, normally present in culture medium and the wound fluid, on the directional migration of human <em>keratinocytes</em> in culture. In electric fields of physiologic strength (100 mV per mm), <em>keratinocytes</em> migrated directionally towards the cathode at a rate of about 1 microm per min. This directional migration requires several <em>growth</em> <em>factors</em>. In the absence of these <em>growth</em> <em>factors</em>, the cell migration rate decreased but directionality was maintained. Epidermal <em>growth</em> <em>factor</em> alone restored cell migration rates at concentrations as low as 0.<em>2</em> ng per ml. Insulin at 5-100 microg per ml or bovine pituitary extract at 0.<em>2</em>%-<em>2</em>% vol/vol also stimulated <em>keratinocyte</em> motility but was not sufficient to fully restore the migration rate. <em>Keratinocyte</em> migration in electric fields requires extracellular calcium. Changes in calcium concentrations from 3 microM to 3.3 mM did not significantly change <em>keratinocyte</em> migration rate nor directionality in electric fields; however, addition of the chelator ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to migration medium reduced, and eventually abolished, <em>keratinocyte</em> motility. Our results show that (i) <em>growth</em> <em>factors</em> and extracellular calcium are required for electric field-induced directional migration of human <em>keratinocytes</em>, and (ii) <em>keratinocytes</em> migrate equally well in low and high calcium media.

Nerve <em>growth</em> <em>factor</em> (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the <em>keratinocyte</em>, a major cellular constituent of the skin, is known to produce other <em>growth</em> <em>factors</em> and cytokines, our experiments were designed to determine whether <em>keratinocytes</em> are a source of NGF. <em>Keratinocyte</em>-conditioned media from the <em>keratinocyte</em> cell line PAM <em>2</em>1<em>2</em> contained NGF-like activity, approximately <em>2</em>-3 ng/ml, as detected by the neurite out<em>growth</em> assay. Freshly isolated BALB/c <em>keratinocytes</em> contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM <em>2</em>1<em>2</em> and BALB/c <em>keratinocytes</em>. Since ultraviolet radiation (UV) is a potentially important modulating <em>factor</em> for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of <em>keratinocyte</em> NGF mRNA (4 h), by <em>2</em>4 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, <em>keratinocytes</em> synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.

Bryostatin 1 and phorbol-1<em>2</em>-myristate-13-acetate (PMA) are both potent activators of protein kinase C (PKC), although in primary mouse <em>keratinocytes</em> bryostatin 1 does not induce differentiation and blocks PMA-induced differentiation. We report here that in primary mouse <em>keratinocytes</em> PMA caused translocation of PKC-epsilon to the Triton X-100-soluble fraction with an approximately <em>2</em>-order of magnitude higher potency, compared with translocation of PKC-alpha and PKC-delta. The kinetics of translocation were fastest for PKC-epsilon, slower for PKC-alpha, and slowest for PKC-delta. At 5-<em>2</em>0 min bryostatin 1 showed potency similar to that of PMA for translocating PKC-alpha, higher potency for translocating PKC-delta, and lower potency for translocating PKC-epsilon. At a later time (6 hr), bryostatin 1 was 1-<em>2</em> orders magnitude more potent than PMA for causing loss of PKC-alpha, -delta, and -epsilon from the soluble fraction. Bryostatin 1 was 40-fold more potent than PMA for down-regulating PKC-alpha and showed a biphasic dose-response curve for down-regulating PKC-delta. Bryostatin 1 at 0.1-1 nM down-regulated PKC-delta to a similar extent as did PMA. Bryostatin 1 at 100 nM to 1 microM, on the other hand, failed to induce down-regulation, and these high (100 nM to 1 microM) doses of bryostatin 1 showed noncompetitive inhibition of PKC-delta down-regulation by 1 microM PMA after coapplication. This protected portion of PKC-delta retained kinase activity. The dose-response curve for bryostatin 1 protection of PKC-delta from down-regulation by PMA correlated with bryostatin 1 inhibition of the effects of PMA on cornified envelope formation (a marker of differentiation) and epidermal <em>growth</em> <em>factor</em> binding. Although PKC-epsilon was readily translocated by both PMA and bryostatin 1, the PKC-epsilon originally associated with the particulate fraction showed no down-regulation by either of these agents. We hypothesize that differential regulation of PKC isozymes by PMA and bryostatin 1 may contribute to the different patterns of biological responses that they induce.

One of the most common side effects of treatment with cyclosporin A (CsA) is hypertrichosis. This study shows that calcineurin activity is associated with hair <em>keratinocyte</em> differentiation in vivo, affecting nuclear <em>factor</em> of activated T cells (NFAT1) activity in these cells. Treatment of nude or C57BL/6 depilated normal mice with CsA inhibited the expression of <em>keratinocyte</em> terminal differentiation markers associated with catagen, along with the inhibition of calcineurin and NFAT1 nuclear translocation. This was associated with induction of hair <em>growth</em> in nude mice and retardation of spontaneous catagen induction in depilated normal mice. Furthermore, calcineurin inhibition blocked the expression of p<em>2</em>1(waf/cip1) and p<em>2</em>7(kip1), which are usually induced with differentiation. This was also associated with an increase in interleukin-1alpha expression (nude mice), a decrease in transforming <em>growth</em> <em>factor</em>-beta (nude and normal mice), and no change in <em>keratinocyte</em> <em>growth</em> <em>factor</em> expression in the skin. Retardation of catagen in CsA-treated mice was accompanied by significant alterations in apoptosis-related gene product expression in hair follicle <em>keratinocytes</em>. The ratio of the anti-apoptotic Bcl-<em>2</em> to proapoptotic Bax expression increased, and expression of p53 and interleukin-1beta converting enzyme activity decreased. These data provide the first evidence that calcineurin is functionally active in follicular <em>keratinocytes</em> and that inhibition of the calcineurin-NFAT1 pathway in these cells in vivo by CsA enhances hair <em>growth</em>.